Fabrication and Use of High-Speed, Concentric H+- and Ca2+-Selective Microelectrodes Suitable for In Vitro Extracellular Recording

2006 ◽  
Vol 96 (2) ◽  
pp. 919-924 ◽  
Author(s):  
Nataliya Fedirko ◽  
Nataliya Svichar ◽  
Mitchell Chesler
Keyword(s):  
Response Time ◽  
High Speed ◽  
Hippocampal Slices ◽  
Brain Slices ◽  
Extracellular Recording ◽  
Neurophysiological Studies ◽  
Sensitivity And Selectivity ◽  
Glass Drawing ◽  
Susceptibility To Noise

Ion-selective microelectrodes (ISMs) have been used extensively in neurophysiological studies. ISMs selective for H+ and Ca2+ are notable for their sensitivity and selectivity, but suffer from a slow response time, and susceptibility to noise because of the high electrical resistance of the respective ion exchange cocktails. These drawbacks can be overcome by using a “coaxial” or “concentric” inner micropipette to shunt the bulk of the ion exchanger resistance. This approach was used decades ago to record extracellular [Ca2+] transients in cat cortex, but has not been subsequently used. Here, we describe a method for the rapid fabrication of concentric pH- and Ca2+-selective microelectrodes useful for extracellular studies in brain slices or other work in vitro. Construction was simplified compared with previous implementations, by using commercially available, thin-walled borosilicate glass, drawing an outer barrel with a rapid taper (similar to a patch pipette), and by use of a quick and reliable silanization procedure. Using a piezoelectric stepper to effect a rapid solution change, the response time constants of the concentric pH and Ca2+-electrodes were 14.9 ± 1.3 and 5.3 ± 0.90 ms, respectively. Use of these concentric ISMs is demonstrated in rat hippocampal slices. Activity-dependent, extracellular pH, and [Ca2+] transients are shown to arise two- to threefold faster, and attain amplitudes two- to fourfold greater, when recorded by concentric versus conventional ISMs. The advantage of concentric ISMs for studies of ion transport and ion diffusion is discussed.

scholarly journals Ginkgo Extract EGb761 Confers Neuroprotection by Reduction of Glutamate Release in Ischemic Brain

2012 ◽  
Vol 15 (1) ◽  
pp. 94 ◽  
Author(s):  
Alexander Mdzinarishvili ◽  
Rachita K. Sambria ◽  
Dorothee Lang ◽  
Jochen Klein
Keyword(s):  
Glutamate Release ◽  
Hippocampal Slices ◽  
Brain Slices ◽  
Cell Swelling ◽  
Experimental Stroke ◽  
Cell Degeneration ◽  
Edema Formation ◽  
Ginkgo Extract

Purpose - Ginkgo extract EGb761 has shown anti-edema and anti-ischemic effects in various experimental models. In the present study, we demonstrate neuroprotective effects of EGb761 in experimental stroke while monitoring brain metabolism by microdialysis. Methods - We have used oxygen-glucose deprivation in brain slices in vitro and middle cerebral artery occlusion (MCAO) in vivo to induce ischemia in mouse brain. We used microdialysis in mouse striatum to monitor extracellular concentrations of glucose and glutamate. Results - In vitro, EGb761 reduced ischemia-induced cell swelling in hippocampal slices by 60%. In vivo, administration of EGb761 (300 mg/kg) reduced cell degeneration and edema formation after MCAO by 35-50%. Immediately following MCAO, striatal glucose levels dropped to 25% of controls, and this reduction was not significantly affected by EGb761. Striatal glutamate levels, in contrast, increased 15-fold after MCAO; after pretreatment with EGb761, glutamate levels only increased by 4-5fold. Conclusions - We show that pretreatment with EGb761 strongly reduces cellular edema formation and neurodegeneration under conditions of ischemia. The mechanism of action seems to be related to a reduction of excitotoxicity, because ischemia-induced release of glutamate was strongly suppressed. Ginkgo extracts such as EGb761 may be valuable to prevent ischemia-induced damage in stroke-prone patients. This article is open to POST-PUBLICATION REVIEW. Registered readers (see “For Readers”) may comment by clicking on ABSTRACT on the issue’s contents page.

Effects of low concentrations of 4-aminopyridine on CA1 pyramidal cells of the hippocampus

1989 ◽  
Vol 61 (5) ◽  
pp. 953-970 ◽  
Author(s):  
P. Perreault ◽  
M. Avoli
Keyword(s):  
High Frequency ◽  
Hippocampal Slices ◽  
Extracellular Recording ◽  
Pyramidal Cells ◽  
Input Resistance ◽  
Peak Latency ◽  
Excitatory Postsynaptic Potential ◽  
Average Value ◽  
Bath Application

1. Intracellular and extracellular recording techniques were used to study the effects of bath application of 4-aminopyridine (4-AP) on pyramidal cells of the CA1 subfield of rat hippocampal slices maintained in vitro. The concentration of 4-AP used in most experiments was 50 microM. However, similar results were obtained with a concentration ranging from 5 to 100 microM. 2. Following 4-AP application, cells impaled with K-acetate-filled microelectrodes hyperpolarized by an average of 2.6 mV (from -68.7 to -71.3 mV, P less than or equal to 0.01). This change was accompanied by the appearance of high-frequency spontaneous hyperpolarizations. Conversely, when KCl-filled microelectrodes were used, an average depolarization of 5.8 mV [from -73.1 to -67.3 mV, not significant (NS)] associated with the occurrence of repetitive depolarizing potentials was observed. In both cases, these changes were concomitant with a small decrease in membrane input resistance, which was statistically significant only for cells impaled with K-acetate-filled microelectrodes. When synaptic transmission was blocked by tetrodotoxin (TTX), 4-AP induced in cells studied with K-acetate microelectrodes an average depolarization of 2.4 mV (from -62.8 to -60.4 mV, P less than or equal to 0.01) accompanied by a small increase in input resistance (from 32.0 to 35.8 M omega, P less than or equal to 0.05). High-frequency spontaneous potentials failed to occur under these conditions. During 4-AP application, the threshold and the latency of action potentials elicited by a depolarizing current pulse increased in 36% of the neurons studied (n = 14). 3. The amplitude of the stratum (s.) radiatum-induced excitatory postsynaptic potential (EPSP) was augmented by 4-AP. Both the early and late inhibitory postsynaptic potentials (IPSPs) evoked by orthodromic stimuli were also increased in amplitude and duration. In addition, a late (peak latency, 150-600 ms) and long-lasting (duration, 600-1,500 ms) depolarizing potential appeared between the early and the late IPSPs and progressively increased until it partially masked these hyperpolarizations. This long-lasting depolarization (LLD) could also be induced by antidromic stimulation, although in this case it was preceded by an additional, fast-rising, brief depolarization. 4. A similar brief depolarization preceded the orthodromically induced LLD in 69% of the neurons bathed in the presence of 4-AP. The average value of the peak latency of this potential was 62 +/- 27 (SD) ms for orthodromic and 110 +/- 70 ms for antidromic responses.(ABSTRACT TRUNCATED AT 400 WORDS)

Physiology and pharmacology of epileptiform activity induced by 4-aminopyridine in rat hippocampal slices

1991 ◽  
Vol 65 (4) ◽  
pp. 771-785 ◽  
Author(s):  
P. Perreault ◽  
M. Avoli
Keyword(s):  
Mossy Fiber ◽  
Epileptiform Activity ◽  
Hippocampal Slices ◽  
Extracellular Recording ◽  
Neuronal Membrane ◽  
Frequency Of Occurrence ◽  
Receptor Antagonists ◽  
Postsynaptic Potentials ◽  
Ca3 Neurons

1. Conventional intracellular and extracellular recording techniques were used to investigate the physiology and pharmacology of epileptiform bursts induced by 4-aminopyridine (4-AP, 50 microM) in the CA3 area of rat hippocampal slices maintained in vitro. 2. 4-AP-induced epileptiform bursts, consisting of a 25-to 80-ms depolarizing shift of the neuronal membrane associated with three to six fast action potentials, occurred at the frequency of 0.61 +/- 0.29 (SD)/s. The bursts were generated synchronously by CA3 neurons and were triggered by giant excitatory postsynaptic potentials (EPSPs). A second type of spontaneous activity consisting of a slow depolarization also occurred but at a lower rate (0.04 +/- 0.2/s). 3. The effects of 4-AP on EPSPs and inhibitory postsynaptic potentials (IPSPs) evoked by mossy fiber stimulation were studied on neurons impaled with a mixture of K acetate and 2(triethyl-amino)-N-(2,6-dimethylphenyl) acetamide (QX-314)-filled microelectrodes. After the addition of 4-AP, the EPSP became potentiated and was followed by the appearance of a giant EPSP. This giant EPSP completely obscured the early IPSP recorded under control conditions and inverted at -32 +/- 3.9 mV (n = 4), suggesting that both inhibitory and excitatory conductances were involved in its generation. IPSPs evoked by Schaffer collateral stimulation increased in amplitude and duration after 4-AP application. 4. The spontaneous field bursts and the stimulus-induced giant EPSP induced by 4-AP were not affected by N-methyl-D-aspartate (NMDA) receptor antagonists 3-3 (2-carboxy piperazine-4-yl) propyl-1-phosphonate (CPP) and DL-2-amino-5-phosphonovalerate (APV) but were blocked by quisqualate/kainate receptor antagonists 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and 6,7-dinitroquinoxaline-2,3-dione (DNQX). CNQX also abolished the presence of small spontaneously occurring EPSPs, thereby disclosing the presence of bicuculline-sensitive (BMI, 20 microM) IPSPs. 5. Small, nonsynchronous EPSPs played an important role in the generation of 4-AP-induced epileptiform activity. 1) After the addition of 4-AP, small EPSPs appeared randomly on the baseline and then became clustered to produce a depolarizing envelope of irregular shape that progressively formed an epileptiform burst, 2) These small EPSPs were more numerous in the 100 ms period that preceded burst onset. 3) The frequency of occurrence of small EPSPs was positively correlated with the frequency of occurrence of synchronous bursts. 4) Small EPSPs and bursts were similarly decreased after the addition of different concentrations of CNQX (IC50 in both cases of approximately 1.2 microM).(ABSTRACT TRUNCATED AT 400 WORDS)

scholarly journals The role of network architecture in the onset of spontaneous activity

STEMedicine ◽  
2020 ◽  
Vol 1 (1) ◽  
pp. e1 ◽  
Author(s):  
Diletta Pozzi ◽  
Nicolò Meneghetti ◽  
Anjan Roy ◽  
Beatrice Pastore ◽  
Alberto Mazzoni ◽  
...  
Keyword(s):  
Spontaneous Activity ◽  
Network Architecture ◽  
Statistical Tests ◽  
Hippocampal Slices ◽  
Brain Slices ◽  
Gabaergic Neurons ◽  
Neuronal Cultures ◽  
Optical Transients

BACKGROUND: The spontaneous activity of neuronal networks has been studied in in vitro models such as brain slices and dissociated cultures. However, a comparison between their dynamical properties in these two types of biological samples is still missing and it would clarify the role of architecture in shaping networks’ operation. METHODS: We used calcium imaging to identify clusters of neurons co-activated in hippocampal and cortical slices, as well as in dissociated neuronal cultures, from GAD67-GFP mice. We used statistical tests, power law fitting and neural modelling to characterize the spontaneous events observed. RESULTS:  In slices, we observed intermittency between silent periods, the appearance of Confined Optical Transients (COTs) and of Diffused Optical Transients (DOTs). DOTs in the cortex were preferentially triggered by the activity of neurons located in layer III-IV, poorly coincident with GABAergic neurons. DOTs had a duration of 10.2±0.3 and 8.2±0.4 seconds in cortical and hippocampal slices, respectively, and were blocked by tetrodotoxin, indicating their neuronal origin. The amplitude and duration of DOTs were controlled by NMDA and GABA-A receptors. In dissociated cultures, we observed an increased synchrony in GABAergic neurons and the presence of global synchronous events similar to DOTs, but with a duration shorter than that seen in the native tissues. CONCLUSION: We conclude that DOTs are shaped by the network architecture and by the balance between inhibition and excitation, and that they can be reproduced by network models with a minimal number of parameters.

scholarly journals Effects of Delivering Guanidinoacetic Acid or Its Prodrug to the Neural Tissue: Possible Relevance for Creatine Transporter Deficiency

2022 ◽  
Vol 12 (1) ◽  
pp. 85
Author(s):  
Enrico Adriano ◽  
Annalisa Salis ◽  
Gianluca Damonte ◽  
Enrico Millo ◽  
Maurizio Balestrino
Keyword(s):  
Hippocampal Slices ◽  
Brain Slices ◽  
Acid Ethyl Ester ◽  
Acute Administration ◽  
Tissue Viability ◽  
Creatine Transporter ◽  
Guanidinoacetic Acid ◽  
Creatine Transporter Deficiency ◽  
Transporter Deficiency

The creatine precursor guanidinoacetate (GAA) was used as a dietary supplement in humans with no adverse events. Nevertheless, it has been suggested that GAA is epileptogenic or toxic to the nervous system. However, increased GAA content in rodents affected by guanidinoacetate methyltransferase (GAMT) deficiency might be responsible for their spared muscle function. Given these conflicting data, and lacking experimental evidence, we investigated whether GAA affected synaptic transmission in brain hippocampal slices. Incubation with 11.5 μM GAA (the highest concentration in the cerebrospinal fluid of GAMT-deficient patients) did not change the postsynaptic compound action potential. Even 1 or 2 mM had no effect, while 4 mM caused a reversible decrease in the potential. Guanidinoacetate increased creatine and phosphocreatine, but not after blocking the creatine transporter (also used by GAA). In an attempt to allow the brain delivery of GAA when there was a creatine transporter deficiency, we synthesized diacetyl guanidinoacetic acid ethyl ester (diacetyl-GAAE), a lipophilic derivative. In brain slices, 0.1 mM did not cause electrophysiological changes and improved tissue viability after blockage of the creatine transporter. However, diacetyl-GAAE did not increase creatine nor phosphocreatine in brain slices after blockage of the creatine transporter. We conclude that: (1) upon acute administration, GAA is neither epileptogenic nor neurotoxic; (2) Diacetyl-GAAE improves tissue viability after blockage of the creatine transporter but not through an increase in creatine or phosphocreatine. Diacetyl-GAAE might give rise to a GAA–phosphoGAA system that vicariates the missing creatine–phosphocreatine system. Our in vitro data show that GAA supplementation may be safe in the short term, and that a lipophilic GAA prodrug may be useful in creatine transporter deficiency.

Ascorbic acid, but not glutathione, is taken up by brain slices and preserves cell morphology

1994 ◽  
Vol 71 (4) ◽  
pp. 1591-1596 ◽  
Author(s):  
M. E. Rice ◽  
M. A. Perez-Pinzon ◽  
E. J. Lee
Keyword(s):  
Ascorbic Acid ◽  
Cell Morphology ◽  
Hippocampal Slices ◽  
Brain Slices ◽  
Neuronal Loss ◽  
Pyramidal Cells ◽  
Cresyl Violet ◽  
Intact Tissue

1. We have determined the ascorbic acid (ascorbate) and glutathione (GSH) content of cortical and hippocampal slices from rat brain after prolonged (6h) incubation and have correlated these levels with the histological quality of the slices. Ascorbate and GSH levels in control and sliced tissue were determined by high performance liquid chromatography (HPLC) with electrochemical detection. Cell morphology of incubated slices was compared with that of intact tissue in cresyl violet stained tissue sections. 2. Roughly 70% of tissue ascorbate and GSH was lost from slices during incubation in vitro. Normal in vivo levels of ascorbate (2-3 mumol g-1 tissue wet weight) could be maintained by including 200-400 microM ascorbate (typical extracellular concentration) in the incubation media. By contrast, the loss of GSH could not be prevented by incubation with GSH. 3. The morphology of cells in hippocampal slices incubated under conditions that maintained ascorbate content and compartmentalization were similar to those of intact tissue. Ascorbate protected pyramidal cells in CA1 and CA3 regions of the hippocampus from the degeneration that was seen in slices incubated in ascorbate-free media. 4. These data suggest that loss of endogenous antioxidants may be a major factor in neuronal loss in vitro and support the notion that ascorbate is an endogenous neuroprotective agent.

Picrotoxin-induced epileptiform activity in hippocampus: role of endogenous versus synaptic factors

1984 ◽  
Vol 51 (5) ◽  
pp. 1011-1027 ◽  
Author(s):  
J. J. Hablitz
Keyword(s):  
Refractory Period ◽  
Mossy Fiber ◽  
Epileptiform Activity ◽  
Hippocampal Slices ◽  
Extracellular Recording ◽  
Synaptic Activity ◽  
Stratum Radiatum ◽  
Resting Membrane Potential ◽  
Stimulation Of

Picrotoxin-(PTX) induced epileptiform activity was studied in guinea pig hippocampal slices maintained in vitro, using intra- and extracellular recording techniques. The observed pattern of spontaneous and evoked epileptiform activity was quite complex. Spontaneous epileptiform events originated in the CA3 region and subsequently spread or propagated to CA1. Activation of CA1 could then reactivate CA3. This reverberation of activity was seen also following stimulation of the mossy fiber afferents from the dentate gyrus to CA3. Stimulation of fibers in the stratum radiatum of the CA1 region could trigger, at short latency, epileptiform activity that either was localized in CA1 or also occurred in CA3, with a late secondary discharge in CA1. This is attributed to a backfiring of the Schaffer collaterals and illustrates the ability of a variety of CA3 inputs to trigger epileptiform activity. Bath-applied PTX, at concentrations of 50-200 microM, had no apparent effect on the resting membrane potential or input resistance of the CA3 cells tested. Depolarizing current pulses elicited characteristic endogenous-burst responses that were not altered by PTX. Synaptic activity evoked by mossy fiber stimulation was altered markedly by PTX. The pattern of observed changes indicated that PTX reduced inhibitory postsynaptic potential (IPSP) amplitudes, resulting in the appearance of repetitive (presumably recurrent) excitatory inputs. Paroxysmal depolarizing shifts ( PDSs ) were generated by the coalescence of these excitatory inputs. Two types of spontaneous bursting were observed after PTX application. The first type was nonepileptiform , all or none in nature, and its frequency was voltage dependent. The second type of spontaneous burst was the PDS. It was epileptiform in character because it was associated with the synchronous discharge of many neurons. It was graded in nature, and its frequency was voltage independent. The graded nature of the PDS was demonstrated by varying the duration and intensity of the orthodromic stimulation. Trains of stimulation could produce PDSs that lasted 500-800 ms. A refractory period was observed following a PDS. By varying the strength of the orthodromic stimulation, it was possible to demonstrate that for the intervals tested this was a relative, not absolute, refractory period. Intracellular recordings in CA3 neurons indicated that each spontaneous PDS was followed by an afterhyperpolarization (AHP).

Na+ and K+ Concentrations, Extra- and Intracellular Voltages, and the Effect of TTX in Hypoxic Rat Hippocampal Slices

2000 ◽  
Vol 83 (2) ◽  
pp. 735-745 ◽  
Author(s):  
Michael Müller ◽  
George G. Somjen
Keyword(s):  
Pyramidal Neurons ◽  
Hippocampal Slices ◽  
Brain Slices ◽  
Input Resistance ◽  
Relative Volume ◽  
Loss Of Function ◽  
Ca1 Region ◽  
Extracellular Compartment ◽  
Membrane Changes

Severe hypoxia causes rapid depolarization of CA1 neurons and glial cells that resembles spreading depression (SD). In brain slices in vitro, the SD-like depolarization and the associated irreversible loss of function can be postponed, but not prevented, by blockade of Na+ currents by tetrodotoxin (TTX). To investigate the role of Na+ flux, we made recordings from the CA1 region in hippocampal slices in the presence and absence of TTX. We measured membrane changes in single CA1 pyramidal neurons simultaneously with extracellular DC potential ( V o) and either extracellular [K+] or [Na+]; alternatively, we simultaneously recorded [Na+]o, [K+]o, and V o. Confirming previous reports, early during hypoxia, before SD onset, [K+]o began to rise, whereas [Na+]o still remained normal and V o showed a slight, gradual, negative shift; neurons first hyperpolarized and then began to gradually depolarize. The SD-like abrupt negative Δ V ocorresponded to a near complete depolarization of pyramidal neurons and an 89% decrease in input resistance. [K+]oincreased by 47 mM and [Na+]o dropped by 91 mM. Changes in intracellular Na+ and K+concentrations, estimated on the basis of the measured extracellular ion levels and the relative volume fractions of the neuronal, glial, and extracellular compartment, were much more moderate. Because [Na+]o dropped more than [K+]o increased, simple exchange of Na+ for K+ cannot account for these ionic changes. The apparent imbalance of charge could be made up by Cl− influx into neurons paralleling Na+ flux and release of Mg2+ from cells. The hypoxia-induced changes in interneurons resembled those observed in pyramidal neurons. Astrocytes responded with an initial slow depolarization as [K+]o rose. It was followed by a rapid but incomplete depolarization as soon as SD occurred, which could be accounted for by the reduced ratio, [K+]i/[K+]o. TTX (1 μM) markedly postponed SD, but the SD-related changes in [K+]o and [Na+]owere only reduced by 23 and 12%, respectively. In TTX-treated pyramidal neurons, the delayed SD-like depolarization took off from a more positive level, but the final depolarized intracellular potential and input resistance were not different from control. We conclude that TTX-sensitive channels mediate only a fraction of the Na+influx, and that some of the K+ is released in exchange for Na+. Even though TTX-sensitive Na+ currents are not essential for the self-regenerative membrane changes during hypoxic SD, in control solutions their activation may trigger the transition from gradual to rapid depolarization of neurons, thereby synchronizing the SD-like event.

scholarly journals New Insights and Methods for Recording and Imaging Spontaneous Spreading Depolarizations and Seizure-Like Events in Mouse Hippocampal Slices

2021 ◽  
Vol 15 ◽  
Author(s):  
Yi-Ling Lu ◽  
Helen E. Scharfman
Keyword(s):  
Dentate Gyrus ◽  
Optical Imaging ◽  
Hippocampal Slices ◽  
Extracellular Recording ◽  
Brain Regions ◽  
Optical Signal ◽  
Artificial Cerebrospinal Fluid ◽  
Acute Hippocampal Slices ◽  
Rats And Mice

Spreading depolarization (SD) is a sudden, large, and synchronous depolarization of principal cells which also involves interneurons and astrocytes. It is followed by depression of neuronal activity, and it slowly propagates across brain regions like cortex or hippocampus. SD is considered to be mechanistically relevant to migraine, epilepsy, and traumatic brain injury (TBI), but there are many questions about its basic neurophysiology and spread. Research into SD in hippocampus using slices is often used to gain insight and SD is usually triggered by a focal stimulus with or without an altered extracellular buffer. Here, we optimize an in vitro experimental model allowing us to record SD without focal stimulation, which we call spontaneous. This method uses only an altered extracellular buffer containing 0 mM Mg2+ and 5 mM K+ and makes it possible for simultaneous patch and extracellular recording in a submerged chamber plus intrinsic optical imaging in slices of either sex. We also add methods for quantification and show the quantified optical signal is much more complex than imaging alone would suggest. In brief, acute hippocampal slices were prepared with a chamber holding a submerged slice but with flow of artificial cerebrospinal fluid (aCSF) above and below, which we call interface-like. As soon as slices were placed in the chamber, aCSF with 0 Mg2+/5 K+ was used. Most mouse slices developed SD and did so in the first hour of 0 Mg2+/5 K+ aCSF exposure. In addition, prolonged bursts we call seizure-like events (SLEs) occurred, and the interactions between SD and SLEs suggest potentially important relationships. Differences between rats and mice in different chambers are described. Regarding optical imaging, SD originated in CA3 and the pattern of spread to CA1 and the dentate gyrus was similar in some ways to prior studies but also showed interesting differences. In summary, the methods are easy to use, provide new opportunities to study SD, new insights, and are inexpensive. They support previous suggestions that SD is diverse, and also suggest that participation by the dentate gyrus merits greater attention.

scholarly journals Extracellular vesicles Transfer Polarized Mitochondria and Increase Cellular Energetics in Ischemic Endothelial Cells

2021 ◽  
Author(s):  
Anisha D’Souza ◽  
Amelia Burch ◽  
Wanzhu Zhao ◽  
Kandarp M. Dave ◽  
Courtney Sabatelle ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Extracellular Vesicles ◽  
Hippocampal Slices ◽  
Brain Slices ◽  
Glucose Deprivation ◽  
List Type ◽  
Biologic Drugs ◽  
Mitochondrial Transfer

AbstractExtracellular vesicles (EVs) such as exosomes (EXOs) and microvesicles (MVs) are promising carriers for the delivery of biologic drugs such as nucleic acids and proteins. We have demonstrated, for the first time, that EVs derived from hCMEC/D3: a human brain endothelial cell (BEC) line transfer polarized mitochondria to recipient BECs in culture and to neurons in mice acute brain cortical and hippocampal slices. This mitochondrial transfer increased ATP levels by 100 to 200-fold (relative to untreated cells) in the recipient BECs exposed to oxygen-glucose deprivation, an in vitro model of cerebral ischemia. Our previous studies suggested that EXOs, the smaller vesicle subpopulation, derived from a macrophage cell line (RAW264.7) load more exogenous plasmid DNA compared to the larger MVs and the RAW-derived EXOs also demonstrated greater transfection in the recipient BECs compared to EXOs derived from the homotypic hCMEC/D3 BECs. Proteomic analysis of EVs indicated that RAW-EVs are preferentially enriched with proteins that are involved in the trafficking of DNA-containing particles from the cytoplasm towards the nucleus. Intriguingly, although the heterotypic macrophage-derived EVs demonstrated increased transfection in the recipient BECs; the homotypic, BEC-derived EVs demonstrated a greater selectivity to transfer polarized mitochondria and increase endothelial cell survival under ischemic conditions.HighlightsEVs transfer polarized mitochondria to endothelial cells and acute brain slicesMitochondrial transfer increased ATP in ischemic brain endothelial cells (BECs)BEC-EVs demonstrate greater mitochondrial transfer to recipient BECsMacrophage-derived EVs are better DNA transfection agents in recipient BECsMacrophage-EVs are enriched in proteins associated with nuclear trafficking of DNA

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